Development of an endoplasmic reticulum calcium sensor based on fluorescence resonance energy transfer

Abstract

Endoplasmic reticulum (ER) is an organelle critically involved in Ca2+ signaling by regulating the intracellular Ca2+ gradient. In the present study, we have developed an ER Ca2+ sensor based on fluorescence resonance energy transfer (FRET), capable of monitoring dynamic Ca2+ signals within the ER. This genetically-encoded ER sensor contains a mutated version of calmodulin (mCaM) and its binding peptide derived from smooth muscle myosin light chain kinase (m-smMLCK) as the Ca2+-responsive elements, thereby enabling an effective ER Ca2+ sensing without perturbation by endogenous CaM. TheCa(2+) sensitivity, including detection limit, of the sensor was evaluated through in vitro characterization. In cell-based assay, our biosensor displayed high accuracy to ER Ca2+ sensing in response to pharmacological stimuli, indicating that this sensor is reliable for visualization of ER Ca2+ signals at high spatiotemporal resolutions in live cells. (C) 2017 Elsevier B.V. All rights reserved.